Mixed function oxidase (MFO) systems mainly consist of three components: cytochrome P-450 (P-450), NADPH-P-450 reductase and phospholipid. With several P-450s, including 2C6, 2CII, 2B4 and 2EI, cytochrome b5 (b5) may participate in the monooxidase pathway by serving in electron transfer from NADPH cytochrome P-450 reductase to P-450. In order to study the functional role of each P-450s and b5, we are preparing a library of monoclonal antibodies (MAbs) to P-450 and b5. Female Balb/c mice were immunized with chemically purified rabbit b5 and the chemically synthesized peptide of N-terminal eight amino acid residues (Lys-Asp-Lys-Glu-Ser-His-Thr-Ala) of rat liver microsomal P-450 3AI as immunogens. Thirteen hybridomas were generated by the fusion of myelomas with spleen cells from mice immunized with rabbit b5. Six of the hybridomas produced IGGI types of MAbs and seven hybridimas produced IgM types. Six hybridomas were generated by the fusion between the myeloma cells and spleen cells from the mice immunized with the peptides. All of the MAbs in culture fluids bound to the immunogens 10 - 20 times greater than control MAbs. The subtypes of MAbs were IGGI, IgG2a and IgM. The MAbs to b5 would be useful for the study of b5 function in MFO systems and the MAbs to P-450 3Al peptide would be more specific in distinguishing subgroups of the P-450 3 family. These MAbs also will be useful for the identification, quantification, and purification of their immunogens in animal and human tissues.